The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, proteintargeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IB␣ phosphopeptide that is recognized by the F-box protein ␤-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF ␤-TRCP , ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin- F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF␤-TRCP -binding phosphopeptide derived from IB␣ linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the ␣ isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.

MUC5AC and MUC5B were specific makers for non-TRU adenocarcinoma, including both central type adenocarcinoma and mucinous adenocarcinoma. We suggest that non-TRU type adenocarcinoma presents a poorer prognosis, so it should be regarded separately from TRU type adenocarcinoma.

The natural product withaferin A (WFA) is a potent angiogenesis inhibitor and it targets the ubiquitin-proteasome pathway in vascular endothelial cells. We generated a biotinylated affinity analog WFA-LC 2 B for use as a probe to study angiogenesis. WFA-LC 2 B inhibits angiogenic sprouting in vitro and it causes levels of ubiquitinated proteins to increase in tumor necrosis factor-α-treated human umbilical vein endothelial cells, confirming the retention of WFA's biological activity. We show that WFA-LC 2 B forms protein adducts in endothelial cells which are competed by free WFA in vivo. This WFA-LC 2 B analog will be useful to isolate the biological target of WFA. KeywordsBiotinylated analog; Natural product; Binding protein; Ubiquitin; Angiogenesis inhibitor Withaferin A (WFA), an important prototype of the withanolide class of natural products ( Fig. 1), is a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera and its related solanaceas species. 1 The withanolides are known to exert very potent and diverse cytotoxic, anti-stress, cardioactive, central nervous system, and immunomodulatory activities. 2 Since the early discovery of WFA during the 1960s, the major interest has been on its anti-tumor cytotoxic activities. 3,4 However, the non-cytotoxic anti-inflammatory 5 and immunomodulatory mechanisms 6 of WFA have thus far remained rather poorly characterized. These latter disease-altering activities are highly pertinent to the practice of ayurveda, a traditional form of Indian medicine, which has borne out many effective formulations from W. somnifera, especially for the treatment of chronic human diseases such as arthritis and female bleeding disorders. 2 Angiogenesis, which is the growth of new blood vessels from preexisting vasculature, is a pathogenic manifestation in cancers, 7 and it is also widely recognized to be critically involved in the pathogenesis of arthritis, endometriosis, age-related macular degeneration, diabetic retinopathy, etc. 7 Since these non-malignant inflammatory diseases could also benefit from anti-angiogenic therapeutics, 8 that such extracts could possess heretofore unrecognized inhibitors of angiogenesis. In fact, we demonstrated that W. somnifera extracts containing non-cytotoxic levels of withanolides, and also WFA, the derived active principle of these extracts, exert potent anti-angiogenic activity in vivo at very low doses. 10 Furthermore, at low nanomolar concentrations, we showed that WFA directly targets endothelial cell proliferation and exerts cytostatic cell cycle G 1 arrest in human umbilical vein endothelial cells (HUVECs). Interestingly, noncytotoxic sub-to-low micromolar concentrations of WFA also inhibit in vitro vessel formation 10,11 in the three-dimensional endothelial cell sprouting assay (3D-ECSA). At such doses, WFA potently inhibits TNF-α-induced NF-κB-DNA-binding activity, a mechanism which is associated with stabilization of phosphorylated IκB-α in the cytoplasm. Our findings suggest that WFA does not in...

Carfilzomib (CFZ) is a second-generation proteasome inhibitor drug approved for the treatment of multiple myeloma. Contrary to its excellent antimyeloma activity, CFZ has shown only limited efficacy in patients with solid malignancies. This lack of efficacy has been attributed in part to rapid degradation of CFZ in the body, possibly hindering the ability of CFZ to access the proteasome target in solid tumors. We hypothesized that polymer micelles, a currently Food and Drug Administration-approved nanoparticle drug delivery formulation, may protect CFZ from metabolic degradation and thus expand the clinical utility of the drug as an anticancer agent. To test our hypothesis, we prepared CFZ-entrapped polymer micelle particles with various compositions and drug release profiles and examined the extent of the CFZ metabolism in vitro using mouse liver homogenates. We also assessed the cytotoxic activities of the CFZ-entrapped micelle formulations in human cancer cell lines derived from B lymphocytes (RPMI-8226) and the lung (H460). Our data indicated that polymer micelle-based formulations can improve metabolic stability and cytotoxic effects of CFZ compared with free CFZ in human cancer cell lines tested. Taken together, these results suggest that polymer micelles may have potential as a delivery system for CFZ with an extended therapeutic utility for nonhematologic malignancies in the future.

Background: The addition of proteasome inhibitors bortezomib and carfilzomib to the multiple myeloma therapy has drastically improved the outcome of patients. However, drug resistance remains a major obstacle in proteasome inhibitor therapy. Several resistance mechanisms have been proposed in vitro, but have not been identified as contributing factors to proteasome inhibitor resistance observed in the clinic. Thus, we set out to investigate previously unexplored resistance mechanisms using BxPC3 pancreatic cancer cells with acquired resistance to carfilzomib. Methods and Results: We established carfilzomib-resistant BxPC3 cells (BxPC3/Cfz) by adapting the parental BxPC3 (human pancreatic cancer) cells to escalating drug concentrations over time. Using cell viability assays, we confirmed that BxPC3/Cfz cells have an increased IC50 values (10-fold) for carfilzomib compared to its parental control. The proteasome activity of resistant and parental BxPC3 cells was measured in vitro using the fluorogenic peptide substrate Suc-LLVY-AMC. Our results showed that majority of the proteasome activity in BxPC3/Cfz cells was restored within 96 hours following initial drug treatment. Decreased drug permeability and enhanced metabolism were ruled out as potential contributors to the lack of activity inhibition. This was based on the lack of efflux transporter upregulation or differences in the rate of drug degradation in the resistant cells compared to parental controls. Given that the recovery of proteasome activity in BxPC3/Cfz cells is much faster than the reported proteasome half-life of 5-12 days, we further examined whether accelerated recovery of proteasome activity in BxPC3/Cfz cells is associated with the development of acquired resistance. To monitor the rate of proteasome degradation, we utilized an activity-based probe to label proteasomes inside cells and in-gel fluorescence assays for visualization. Using a similar approach, we also measured the rate of proteasome synthesis by western blotting analysis of unlabeled proteasome subunits. Conclusion: Here, we report that the acquired resistance to carfilzomib in BxPC3 cells does not involve previously reported resistance mechanisms, but may arise from accelerated proteasome turnover. In order to further validate our findings as a novel mechanism determining cellular sensitivity to proteasome inhibition, we are currently assessing whether cancer cells that are intrinsically resistant to proteasome inhibition have increased rates of proteasome turnover compared to sensitive cells. Taken together, our findings may provide previously unaddressed insights into cancer resistance to proteasome inhibition. Citation Format: Lin Ao, Jieun Park, Di Hu, Hyun Young Jeong, Kyung Bo Kim, Wooin Lee. Accelerated proteasome turnover as a potential mechanism of acquired resistance to carfilzomib in BxPC3 pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1707. doi:10.1158/1538-7445.AM2014-1707

No abstract